Lobetyolin Alleviates Ferroptosis of Skeletal Muscle in 5/6 Nephrectomized Mice via Activation of Hedgehog-GLI1 Signaling.

BACKGROUND: Muscle wasting increases morbidity and mortality and is related to chronic kidney disease (CKD) and dialysis. It is still unclear whether ferroptosis occurs during this progression and whether it is a potential intervention target for the treatment of CKD-related muscle injury. PURPOSE: The objective is to identify potential compounds for treating ferroptosis and muscle wasting and explore the potential mechanisms in vivo/in vitro. METHODS: Initially, we explored whether ferroptosis is present in the skeletal muscle of 5/6 nephrectomized (NPM) mice via RNA-Seq analysis, TUNEL staining, Oil red O staining, MDA/GSH/GSSG level detection and real-time quantitative PCR (qPCR). Subsequently, utilizing our established molecular phenotyping strategy, we screened potential traditional Chinese herb-derived compounds for alleviation of muscle wasting and ferroptosis. HE staining, Oil red O staining, TUNEL staining, immunofluorescence staining, MDA/GSH/GSSG level detection, Fe level detection, western blotting and qPCR were applied to assess the effects of the identified compound on muscle wasting and ferroptosis and explore the potential mechanism. Furthermore, RNA-Seq analysis, ChIP-Seq analysis and further experiments in vitro were performed to determine the role of Hedgehog signaling in the effect of Lobetyolin (LBT) on ferroptosis. RESULTS: In NPM mice, skeletal muscle dysfunction, lipogenesis, reduced GSH/GSSG ratio, decreased GSH content, increased MDA production and and higher levels of ferroptosis markers were observed. LBT treatment (30 mg/kg or 50 mg/kg) significantly alleviates skeletal muscle injury by inhibiting ferroptosis. Additionally, in an in vitro investigation, C2C12 cells exposed to Indolyl sulfate (IS) induced ferroptosis and LBT treatment (20 µM and 50 µM) protected C2C12 from such injury, consistent with the results from the in vivo analysis. Furthermore, it was found LBT increased the levels of protein involving Hedgehog signaling pathway (SMO and GLI1), and rescue analysis revealed that this pathway played a crucial role in the regulation of ferroptosis. Further experiments demonstrated that LBT upregulated a series of suppressors of ferroptosis by activating Gli1 transcription. CONCLUSION: LBT alleviates CKD-induced muscle injury by inhibiting ferroptosis through activation of the Hedgehog signaling pathway.

Sclederma of Poria cocos exerts its diuretic effect via suppression of renal aquaporin-2 expression in rats with chronic heart failure.

ETHNOPHARMACOLOGICAL RELEVANCE: Sclederma of Poria cocos (Hoelen) has been used as a diuretic in traditional Asian medicine. However, the underlying mechanism by which Sclederma of Poria cocos (hoelen) exerts its diuretic effect has not been well identified. The aim of the present study was to evaluate the effects of Sclederma of Poria cocos (hoelen) in rats with chronic heart failure (CHF) induced by acute myocardial infarction and to investigate the underlying mechanisms. MATERIALS AND METHODS: An aqueous extract of Sclederma of Poria cocos (hoelen) (2.4 g/kg/d, 1.2 g/kg/d or 0.6 g/kg/d) or furosemide (20 mg/kg/d) was administered orally to male Sprague-Dawley rats starting on the day of coronary ligation. The urine output of all rats was quantified and collected every day for 1 or 4 weeks. The expression of aquaporin-2 (AQP2) was examined after treatment for 1 or 4 weeks. RESULTS: Urinary output increased significantly and urinary osmolality decreased after oral administration of Sclederma of Poria cocos (hoelen) for both 1 and 4 weeks. Sclederma of Poria cocos (hoelen) caused less electrolyte disorder than furosemide. Furthermore, Sclederma of Poria cocos (hoelen) reduced the levels of plasma BNP in CHF rats, whereas furosemide had no effect. Importantly, both mRNA and protein expression of AQP2 were down-regulated and urinary excretion of AQP2 was decreased after administration of Sclederma of Poria cocos (hoelen) to CHF rats. Similarly, Sclederma of Poria cocos (hoelen) reduced plasma arginine vasopressin (AVP) level and down-regulated vasopressin type 2 receptor (V2R) mRNA expression. CONCLUSIONS: Sclederma of Poria cocos (hoelen) exerts its diuretic effect and improves cardiac function in CHF rats via the AVP-V2R-AQP2 axis.